Infectious Salmon Anemia (ISA) is a viral exclusive in culture disease of the Atlantic salmon (Salmo salar L.), discovered in 1984 in Norway (Thorud and Djupvik, Infectious anemia in Atlantic salmon (Salmo salar L.), Bull. Eur. Assoc. Fish Pathol. 8: 109-111, 1988) and it is characterized by provoking high mortality in fish. The most common signs are severe anemia; exophthalmos; ascites; petechial hemorrhage in viscera, adipose tissue and skin; and hemorrhagic necrosis in the liver of infected fish (Falk and Dannevig, infectious salmon anemia Demonstration of (ISA) viral antigens in cell cultures and tissue sections. Vet. Res 26: 499-504, 1995; Falk et al, Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.), J. Virol. 71: 9016-9023, 1997, Munir and Kibenge, Detection of infectious salmon anemia virus by real-time RT-PCR. J. Virol. Methods 117: 37-47, 2004). In the economic field, this disease has caused great losses in salmon producing countries where it has been declared, such as Norway, Canada, Scotland, Faroe Islands and the United States (Kibenge et al, Isolation and identification of infectious salmon anemia virus (ISAV) from Coho salmon in Chile, Dis Aquat Organ 45, 9-18, 2001; Lovely et al, First identification of infectious salmon anemia virus in North America with haemorrhagic kidney syndrome, Dis Aquat Organ 35, 145-148, 1999; Falk and al, Identification and characterization of viral structural proteins of infectious salmon anemia virus, J Virol 78, 3063-3071, 2004). Moreover, in the case of Norway, the high mortality produced losses covering almost the entire production, reporting up to 80% reductions in the total production of the country. Chile, a country that until recently was free of the disease, has also been attacked by the virus, partly due to the globalization of markets and on the other hand due to the intense production conditions at the farms, which allows an easy dissemination, representing a problem often difficult to control. Finally the pathogen agent was first detected in Chile in 2001 (Kibenge et al, Isolation and identification of infectious salmon anemia virus (ISAV) from Coho salmon in Chile, Dis Aquat Organ 45, 9-18, 2001).
The etiologic agent of this disease is the Infectious Salmon Anemia Virus (ISAV). It belongs to the Orthomyxoviridae family, it has 8 segments of single-stranded genomic RNA of negative polarity, coding for 8 structural proteins and 2 nonstructural proteins. The genomic organization of ISAV has situated it in a new genus, the Isavirus or Aquaorthomyxovirus (Krossoy et al, The putative polymerase sequence of infectious salmon anemia virus Suggests a new genus Within the Orthomyxoviridae, J Virol 73, 2136-2142, 1999). The 8 genomic RNA segments are attached to multiple copies of the viral nucleoprotein (NP), a copy of the RNA polymerase complex formed by proteins PB1, PB2 and PA is situated at the ends 3′; which altogether are called ribonucleoproteins. A membrane envelope in which the glycoproteins Hemagglutinin-esterase (HE) and fusion (F) are inserted surrounds the protean capsid, which is formed by matrix protein M1 and M2, (Aspehaug et al, Characterization of the infectious salmon anemia virus fusion protein, J Virol 79, 12544-12553, 2005).
The ISAV replicative cycle is very similar to the influenza A virus, where the HE protein identifies a cellular receptor containing 4-O-acetyl-sialic acid (Hellebo et al, Infectious salmon anemia virus binds to and hydrolyzes especificamente 4-O-acetylated sialic acids, J Virol 78, 3055-3062, 2004). Subsequently the particle is entered into the cell in vessels covered with clathrin, which are fused to endosomes, providing the necessary acidic environment for the fusion among membranes, both viral and endosomal, allowing the stripping of the virus. The viral ribonucleoproteins are forwarded to the nucleus of the cell, where viral transcription starts. The viral mRNA produced at the nucleus is translated in the cellular cytoplasm, returning to the nucleus only the NP, PB1, PB2 and PA proteins; allowing the beginning of viral replication and subsequently, the formation of ribonucleoproteins. The assembly of mature viral particles is performed in the cell membrane toward which the glycoproteins are headed, HE and F, and also M proteins, which will be the NP receptors, finally releasing the virions by a budding process.
The study of diseases caused by microorganisms allows the design of different control strategies, either with early diagnosis, confronting the spread of the disease or mainly attacking its origin, that is, the same microorganisms. In the case of virus, it is essential to study both the replicative cycle in general and the function and structure of each of the viral proteins. Through these studies it has been found that the orthomyxovirus undergoes a high rate of mutation and recombination forming new strains from one year to another. This is the main reason why prevention through vaccination does not guarantee protection against infection with these agents, leading to develop alternative therapies to prevent the spread of infection within and between organisms. The design and testing of compounds that interfere with the replication cycle of the virus is a relevant area of study for the control of the orthomyxovirus as ISAV.
To date, approximately 40 antiviral compounds have been approved for its use in humans, primarily for the treatment of infections caused by the human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes virus. On the other hand, the number of approved antiviral compounds that can be used for the treatment of infections caused by RNA is limited. Among these are, without considering treatment for HIV, the M2 channel inhibitors, amantadine and rimantadine, and the neuraminidase inhibitors, oseltamivir and zanamivir for influenza; and ribavirin for the treatment of respiratory syncytial virus (RSV), the hepatitis C virus (HCV) and is also being used for the treatment of Lassa fever (Revised In: Leyseen et al, Molecular Strategies to inhibit the replication of RNA viruses, Antivir. Res 78: 9-15, 2008).
Ribavirin is a synthetic nucleoside whose chemical name is 1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide having the following structural formula (Formula I):

Ribavirin is a broad-spectrum inhibitor of RNA virus replication, which has been approved for treatment of HCV infections in combination with interferon and for the treatment of RSV infections in pediatric aerosol form. It has also been used experimentally for other conditions, including Lassa fever (Khan et al, New Opportunities for field research on the pathogenesis and treatment of Lassa fever, Antivir. Res 78: 103-115, 2008), CCHF virus (Ergonul, Treatment of Crimean-Congo hemorrhagic fever. Antivir res 78: 125-131, 2008) and Hantavirus (Jonsson et al. hantavirus pulmonary syndrome Treatment of, Antivir. res 78: 162-169, 2008). While it has been found that the compound inhibits in vitro replication of some viruses studied, most of them being RNA virus, it has been shown to have a protective effect only in some animal models. Also, the power of the in vitro activity of ribavirin may vary considerably depending on the nature of the virus (Graci and Cameron, Mechanism of action of ribavirin against distinct viruses, Rev. Med. Virol. 16, 37-48, 2006).
It has been reported that most of RNA viruses are sensitive to the activity of ribavirin in vitro, but only some of them are more susceptible than others. In general, ribavirin is not very potent as an antiviral medication regularly showing EC50 values (effective concentration 50%) of 1 μM or even higher. Specifically, in vitro antiviral activity has been proved against bunyaviruses including CCHF virus (hemorrhagic fever Crimean-Congo), Rift Valley fever virus, and Hantavirus. It also inhibits the replication of coronaviruses, including coronavirus-SARS and flaviviruses, but has shown not to be effective in animals experimentally infected with these viruses (Watts y cols, Inhibition of Crimean-Congo hemorrhagic fever viral infectivity yields in vitro by ribavirin. Am. J. Trop. Med. Hyg. 41: 581-585, 1989; Huggins, Prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug. Rev. Infect. Dis. 11 (Suppl. 4) S750-S761, 1989; Severson y cols, Ribavirin causes error catastrophe during Hantan virus replication. J. Virol. 77: 481-488, 2003; Saijo y cols, Inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (SARS)-associated coronavirus. Antivir. Res. 66: 159-163, 2005; Barnard y cols, Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin. Antivir. Res. 71: 53-63, 2006; Neyts y cols, Use the yellow fever virus vaccine strain 17D for the study of strategies for the treatment of Bellow fever virus infections. Antivir. Res. 30 (2-3): 125-132, 1996). At a similar way, ribavirin is not effective in animal models infected with filovirus (Huggins, Prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug. Rev. Infect. Dis. 11 (Suppl. 4) S750-S761, 1989). It has also been observed that ribavirin is effective in vitro and in vivo against RSV, a paramyxovirus, and it reveals relative sensitivity in cell cultures of other paramyxoviruses, the Nipah virus, but only limited efficacy in animals models has been observed (Snell, ribavirin therapy for Nipah virus infection. J. Virol. 78: 10211, 2004, Georges-Courbot et al, Poly-(I)-poly (C12U) but not ribavirin prevents it death in a hamster model f Nipah virus infection. Antimicrob. Agents Chemother. 50: 1768-1772, 2006).
It has also been evaluated the effect of ribavirin on VHSV virus (Viral Hemorrhagic Septicemia Virus), in EPC cell cultures (Ephitelioma papulosum cyprini). Cells were infected with the virus and treated with 1 to 25 μg/ml Ribavirin. The results revealed a strong inhibition of the virus at concentrations of 5, 10 and 25 μg/ml. They also show a high inhibition of viral RNA accumulation when 25 μg/ml are added at 0 hours post infection, occurring RNA inhibition of 99.8% at 10 hours post infection. This report concludes that the measuring method employed (RT-PCR in real time) can be used in combination with the classical methods to study the progression of the infection and the kinetics of virus replication, but there is however, not always a correlation of in vitro results with the protection given to the fish, so it is necessary to conduct tests in vivo (Moroccan et al., Assessment of the inhibitory effect of ribavirin on the rainbow trout rhabdovirus VHSV by real-time reverse-transcription PCR, Vet. Microbiol. 122: 52-60, 2007). In this case, a test was conducted with a virus that infects the rainbow trout and its effect in vitro was assayed. There are no reports in vivo and not anything suggests that the same effects on cells in culture could be seen in fish.
On the other hand the antiviral effect of ribavirin in cell cultures infected with IPNV virus (Infectious Pancreatic Necrosis Virus) has been evaluated (Jashés et al. Inhibitors of infectious pancreatic necrosis virus (IPNV) replication, Antiviral Res 29: 309-312, 1996, Hudson et al, The Efficacy of amantadine and other antiviral compounds against salmonid two viruses in vitro, Antiviral Res 9: 379-385, 1988). It was observed that this antiviral is capable of inhibiting viral replication in vitro of the IPN virus with an EC50 of 0.5 g/mL and a CC50 of 100 μg/mL, but it was not the most effective antiviral and the in vivo activity was no longer evaluated (Jashés et al. Inhibitors of infectious pancreatic necrosis virus (IPNV) replication, Antiviral Res 29: 309-312, 1996).
In an in vivo study reported in 1980, the effect of ribavirin on IPNV infected rainbow trout was analyzed. The compound was supplied through a solution in the tank where the fish were held by exposure to the antiviral once during two hours. The administration was performed in increasing concentrations to the fish into two separate batches of 6 tanks each. The results revealed a decreased rate of dead fish in about 5%, but there was not a linear effect over the concentration of the antiviral used. Also the higher dose of ribavirin administered, 400 μg, produced no greater decrease in the rate of death. It is concluded that higher doses of ribavirin do not produce a greater decrease in the quantity of dead fish. An alternative treatment option would be a sustained exposure to antiviral in order to decrease significantly the death rate of infected fish. However it is suggested that the costs involved would be very expensive and most fish farm owners would be hostile to initiate any antiviral treatment before the existence of an apparent viral illness (Sayan and Dobos. Effect of Virazole on rainbow trout fry Salmo gairdneri Richardson infected with infectious pancreatic necrosis virus, J. Fish Dis., 3: 437-440, 1980). No further testing is reported, so that the usefulness of ribavirin in the treatment of IPNV infected trout has not been demonstrated.
There have been several proposals of molecular mechanisms responsible for the antiviral activity of ribavirin (Hong and Cameron, pleiotropic Activities Mechanisms of antiviral ribavirin, Prog Drug Res 59: 41-69, 2002; Revised: Leyseen et al, Molecular Strategies to inhibit the replication of RNA viruses, Antivir. Res 78: 9-15, 2008). These mechanisms include: (1) depletion of intracellular levels of GTP by inhibition of intracellular IMP dehydrogenase caused by 5′-monophosphate metabolite of ribavirin, (2) inhibition of the viral polymerase activity, caused by the 5′-triphosphate metabolite of ribavirin, (3) inhibition of the viral capsid through inhibition of the guaniltransferase activity caused by the 5′-triphosphate ribavirin, (4) inhibition of viral helicase causing the process known as catastrophic error resulting of the accumulation of mutations, some of them being lethal, in the viral genome.
In the present, it is still in debate the magnitude of the contribution of the catastrophic error, of the depletion of intracellular levels of GTP and other proposed mechanisms of antiviral activity of ribavirin, and the way they would contribute in the in vivo activity of this antiviral.
As it has already been mentioned, ribavirin is used in the chronic treatment of hepatitis C in humans, in association with interferon alpha. The information available for the product Rebetol®, containing ribavirin as active ingredient, establishes that the FDA (Food and Drug Administration) alerts about an important primary toxicity of this compound, because it triggers hemolytic anemia in patients treated with the product and can lead to a worsening of cardiac disease causing both fatal and non-fatal myocardial infarction.
From the available information the need for a treatment for fish infected with ISA virus is required. It is in fact a problem in the aquaculture farms, and there is no currently an effective and efficient solution, that would arrest and reverse infections caused by these viruses. If is not arrest, they could cause great economic losses and producers and consumers will be affected significantly.
The fact of the need of an effective and efficient treatment is also related to its cost, so as not to increase the price of the final product, in order to give the possibility to dispense a product not only when the disease has spread but also, and as it has been suggested for other pathologies treated with antiviral drugs, to be able to dispense it in the early stages of infection, thus allowing more protection by reducing the propagation of the replication stage in which the virus is. This would prevent healthy fish from being infected by cohabitation, blocking the spread and eventually stopping the disease.
From the available data, there is not a compound capable of inhibiting the ISA virus and even more that could do it in vivo, or in naturally infected fish.
As it has been mentioned previously, considering the high mutation presented by the ISA virus that leads to a change in vaccines every year because the effectiveness decrease, the problem should be addressed by looking at the properties of the etiologic agent remaining in time and that are vulnerable to other mechanisms. In view of the ISA virus shares certain characteristics with the influenza virus, the inventors concluded that an efficient way to control the disease could be use of known efficient antivirals to inhibit these viruses. But surprisingly, inventors found that out of all antiviral tested, only ribavirin showed to be really effective in vitro.
Ribavirin is a broad-spectrum antiviral compound, but the background and the available data do not suggest that it might be useful for the treatment of this disease, even more considering that it has been reported that one of its most important toxic effects is hemolytic anemia. An expert in the art would never even think about using it for the treatment of an infection caused by ISAV, disease characterized by the development of severe anemia, among other complications, which altogether are ultimately fatal to the fish.
Contradicting all predicted ideas, the inventors of the present invention have surprisingly found that ribavirin is effective in the treatment of fish infected with ISA virus.